1. Field of the Invention
The present invention relates to an improved method for the transfection of filamentous fungi.
2. Description of Related Art
Eukaryotic cells are preferred organisms for the production of polypeptides and secondary metabolites. In fact, filamentous fungi are widely used in large-scale industrial processes as fytase or penicillin production. To stay competitive these fermentation processes need continuous optimization. Classical strain improvement projects are widely used, but deliver only incremental improvements. New technologies as Genomics and Systems Biology in theory should be able to bring significant improvements, but until now no example has been reported for an industrial (fungal) process.
So far, examples were only reported for lab-strains and limited to few genes (Theilgaard et al, 2001, Biotechnol. Bioeng. 72:379-388; Abe et al., 2002, Mol. Genet. Genomics. 268:130-137; Askenazi et al., 2003, Nat. Biotechnol. 21:150-156). Although successful, the results are not leading to any commercial application as the productivities of such strains are several factors lower than industrial production strains. The major problem being the many leads for recombinant and targeted strain improvement coming from, for example, transcriptomics data (see Askenazi et al., 2003). With the relatively large genome size and the low genetic amenability of fungi it has not been possible to study the effect of 100+ gene modifications, let alone the combination of these. This may be the key problem causing this lack of results in industrial (fungal) applications. The average fungal genome consists of 13.000 genes, meaning that considering only the over expression of all genes leads already to 13.000 solutions. If also, deletions and kinetic alterations are considered, this number will increase further.
To decrease this number to a workable number all kinds of ‘omics’ technologies are applied in expensive R&D projects, collected under the name Systems Biology. However, these very huge datasets can only reduce the number of lead genes typically to several hundred. Evaluating all the combinations (say 300!) in practice, i.e. actually modifying the genes and/or expression level, still is quite laborious. Especially, considering the quite tedious transformation procedures needed to transform filamentous fungi. So, for these new technologies to deliver significant improvements in productivity of industrial fungal strains large number of transformants would have to be screened before a transformant with the properties of interest can be isolated. There is thus a need for an efficient transfection method that would enable one to quickly produce and screen many combinations and thereby increase the chance of detecting DNA sequences encoding proteins determining significant increases in process output. Present transfection systems for filamentous fungi are very laborious (see for review Fincham, 1989, Microbiol. Rev. 53:148-170). This involves protoplast formation, viscous liquid handling (i.e. polyethylene glycol solutions), one-by-one swirling of glass tubes and subsequent selective plating. On top of that the efficiency of homologous targeting was until recently very low, resulting in mostly random integrated DNA fragments, which quite often are integrated as multiple tandem repeats (see for example Casqueiro et al., 1999, J. Bacteriol. 181:1181-1188). This uncontrolled “at random multiple integration” of an expression cassette is a potentially dangerous process, which can lead to unwanted modification of the genome of the host. It is therefore highly desirable to be able to construct a production strain by ensuring the targeting of the expression cassette to the right genomic locus with high efficiency. Both technological limitations severely hampered a rapid progress in targeted improvements of industrial fungal processes.
With the current explosion of available genome sequences and a significant improvement of homologous targeting in fungi by disturbing the non-homologous end-joining pathway (see for example Ninomiya et al., 2004, Proc. Natl. Acad. Sci. USA 101:12248-12253) it should be possible to quickly assess gene function in relation to industrial application and construct significantly improved industrial fungal strains. In addition to that also other tools are rapidly developed towards high throughput application, like the GATEWAY cloning system (Invitrogen) and genome wide GFP tagging of proteins (Toews et al., 2004, Curr. Genet. 45:383-389).
However despite those new tools being available for High Throughput functional analysis of genes, methods for High Throughput fungal transfection are lacking, hampering the application of those tools. Recently, several advances or alternatives were reported for fungal transfection methods, including:                Efficient gene targeting and fungal transfection frequencies were obtained after Agrobacterium tumefaciens co-transformation (Michielse et al., 2005, Fungal Genet. Biol. 42:9-19); however this method needs co-cultivation of both species in shake flasks, and is thus not amendable for High Throughput applications.        Genetic transformation by micro projectile bombardment (Aida V. Rodriguez-Tovar et al., 2005, J. Microbiol. Meth. 63:45-54.); this method was less efficient than Agrobacterium-mediated transformation and also involves one Petri-dish per experiment, and is thus not amendable for High Throughput applications.        Electroporation seems a very fast way to transfect species and it was also established for fungi, but only for a limited set of species, like Neurospora crassa (Chakraborty et al., 1991, Can. J. Microbiol. 37:858-863). However, this method requires germination of conidia, resulting in a multi cellular system of which one cell may be transfected. Subsequently, this mixed cell system needs to be colony purified as only transfected cells are wanted, and therefore this method is not amendable for High Throughput applications.        
So, despite many technological developments, there is no efficient and economically attractive procedure to perform high throughput transformations of filamentous fungi available, but as fungi are very important commercial species such a method is extremely desirable.